The present invention is directed toward the development of an oleaginous yeast that accumulates oils enriched in long-chain ω-3 and/or ω-6 polyunsaturated fatty acids (“PUFAs”; e.g., 18:3, 18:4, 20:3, 20:4, 20:5, 22:6 fatty acids). Toward this end, the natural abilities of oleaginous yeast (mostly limited to 18:2 fatty acid production) have been enhanced by advances in genetic engineering, leading to the production of 20:4 (arachidonic acid or “ARA”), 20:5 (eicosapentaenoic acid or “EPA”) and 22:6 (docosahexaenoic acid or “DHA”) PUFAs in transformant Yarrowia lipolytica. These ω-3 and ω-6 fatty acids were produced by introducing and expressing heterologous genes encoding the ω-3/ω-6 biosynthetic pathway in the oleaginous host (see co-pending U.S. patent applications Ser. No. 10/840,579 and Ser. No. 60/624,812, each entirely incorporated herein by reference). However, in addition to developing techniques to introduce the appropriate fatty acid desaturases and elongases into these particular host organisms, it is also necessary to increase the transfer of PUFAs into storage lipid pools following their synthesis.
Most free fatty acids become esterified to coenzyme A (CoA) to yield acyl-CoAs. These molecules are then substrates for glycerolipid synthesis in the endoplasmic reticulum of the cell, where phosphatidic acid and diacylglycerol (DAG) are produced. Either of these metabolic intermediates may be directed to membrane phospholipids (e.g., phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine) or DAG may be directed to form triacylglycerols (TAGs), the primary storage reserve of lipids in eukaryotic cells.
In the yeast Saccharomyces cerevisiae, three pathways have been described for the synthesis of TAGs. First, TAGs are mainly synthesized from DAG and acyl-CoAs by the activity of diacylglycerol acyltransferases. More recently, however, a phospholipid:diacylglycerol acyltransferase has also been identified that is responsible for conversion of phospholipid and DAG to lysophospholipid and TAG, respectively, thus producing TAG via an acyl-CoA-independent mechanism (Dahlqvist et al., PNAS. 97(12): 6487–6492 (2000)). Finally, two acyl-CoA:sterol-acyltransferases are known that utilize acyl-CoAs and sterols to produce sterol esters (and TAGs in low quantities; see Sandager et al., Biochem. Soc. Trans. 28(6):700–702 (2000)).
A comprehensive mini-review on TAG biosynthesis in yeast, including details concerning the genes involved and the metabolic intermediates that lead to TAG synthesis, is that of D. Sorger and G. Daum (Appl. Microbiol. Biotechnol. 61:289–299 (2003)). However, the authors acknowledge that most work performed thus far has focused on Saccharomyces cerevisiae and numerous questions regarding TAG formation and regulation remain. In this organism it has been conclusively demonstrated that only four genes are involved in storage lipid synthesis: ARE1 and ARE2 (encoding acyl-CoA:sterol-acyltransferases), LRO1 (encoding a phospholipid:diacylglycerol acyltransferase, or PDAT enzyme) and DGA1 (encoding a diacylglycerol acyltransferase, or DGAT2 enzyme) (Sandager, L. et al., J. Biol. Chem. 277(8):6478–6482 (2002)).
Although homologs of the acyltransferase genes described above have been identified in various other organisms and disclosed in the public literature, few genes are available from oleaginous organisms. Concerning diacylglycerol acyltransferases, a single DGAT2 enzyme from oleaginous yeast (i.e., Yarrowia lipolytica) has been isolated and characterized in co-pending U.S. patent application Ser. No. 10/882,760. Since the natural capabilities of this organism are limited to 18:2 fatty acid production, however, its native diacylglycerol acyltransferases (including DGAT2) are not likely to utilize longer chain PUFAs (i.e., C20 or greater) as efficiently as those from organisms that are naturally capable of producing longer chain PUFAs. The production of ARA, EPA and DHA PUFAs in transformant Yarrowia lipolytica therefore is likely to be improved by the use of heterologous acyltransferases (e.g., DGAT2) having altered substrate specificies, as compared to the native enzymes. Furthermore, techniques for modifying the transfer of fatty acids to the TAG pool in oleaginous yeast have not been developed.
Only three DGAT2 enzymes have been isolated from oleaginous fungi. Specifically, Lardizabal et al. isolated and characterized two DGAT2s (i.e., MrDGAT2A and MrDGAT2B) from Mortierella ramanniana (J. Biol. Chem. 276(42):38862–28869 (2001)); US 2003/0028923 A1, US 2003/0115632 A1) and one DGAT2 from Neurospora crassa (Nc DGAT2; see US 2004/0107459 A1). Upon expression of MrDGAT2A, MrDGAT2B, and NcDGAT2 in insect cells, high levels of DGAT activity were obtained on membranes isolated from those cells. Like Y. lipolytica, however, M. ramanniana and N. crassa are generally limited to production of 16:0, 18:0, 18:1, 18:2 and 18:3 fatty acids (although synthesis of 20:0 fatty acids is observed in M. ramanniana during lower temperature growth; see da Silva M., et al. Rev. Microbiol. 29:4 São Paulo (October/December 1998)). Thus, the DGAT2s from M. ramanniana and N. crassa are likely not preferred for the transfer of 20:4, 20:5 and 22:6 PUFAs to the TAG pool in a transformant oleaginous yeast.
A variety of microorganisms are known that naturally produce long-chain PUFAs (e.g., ARA and EPA, DHA). For example, microorganisms in the genera Mortierella (filamentous fungus), Entomophthora, Pythium and Porphyridium (red alga) can be used for commercial production of the ω-6 fatty acid, ARA. The fungus Mortierella alpina, for example, is used to produce an oil containing ARA, while U.S. Pat. No. 5,658,767 (Martek Corporation) teaches a method for the production of an oil containing ARA comprising cultivating Pythium insidiuosum in a culture medium containing a carbon and nitrogen source. Likewise, U.S. Pat. No. 5,244,921 (Martek Corporation) describes a process for producing an edible oil containing EPA, by cultivating the heterotrophic diatoms Cyclotella sp. and Nitzschia sp. in a fermentor. DHA can be obtained by cultivation of the heterotrophic microalgae Crypthecodinium cohnii (U.S. Pat. No. 5,492,938 and U.S. Pat. No. 5,407,957). Other long-chain PUFA-producing organisms include Thraustochytrium sp. and the green alga Parietochloris incisa. It is likely that many of these organisms possess genes encoding acyltransferases that would be preferred for the incorporation of long-chain PUFAs in a transformant oleaginous yeast, relative to the native acyltransferases that do not naturally produce long-chain PUFAs.
Thus, there is a need for the identification and isolation of a gene encoding an acyltransferase from an organism such as those above, to permit its use in the production and accumulation of long-chain PUFAs in the storage lipid pools (i.e., TAG fraction) of transformant oleaginous yeast.
Applicants have solved the stated problem by isolating the gene encoding DGAT2 from the oleaginous fungus, Mortierella alpina. This gene will be useful to enable one to modify the transfer of long-chain free fatty acids (e.g., ω-3 and/or ω-6 fatty acids) to the TAG pool in oleaginous yeast.